Effect On Myocardial Microvascular Regeneration By Myocardial Vascular AIBP Knocking Down

BackgroundWith the aging of social population and the improvement of people’s living standard, the coronary heart disease has become common and frequently-occurring which harms human health. The morbidity and death rate show a rising trend year by year, and the age of onset also starts to show younger trend. In terms of the treatment on ischemic heart disease, the primary thing is to recover hemoperfusion, and relieves ischemia, hypoxia, and insufficient supply of nutrient substance with myocardial tissue. The regenerated blood vessel can improve the blood supply of cardiac muscle to a great degree, thus alleviating the progression of disease. The research on how to promote the vascular regeneration and improve microcirculation after myocardial anoxia and ischemia attracts more and more attention. It has become one of important directions researching on ischemic cardiomyopathy therapeutics.It is found via recent research that apo A-I binding protein(AIBP) secreted by peripheral tissues can restrain the angiopoiesis. Therefore, we can theoretically deduce AIBP activity that can stimulate the re-growth of blood vessel. However, until now there is no report on the function of AIBP in cardiac microvascular endothelial cells(CMECs) and cardiac muscle tissue. Therefore we carry out a research on whether it is able to stimulate cardiovascular regeneration after myocardial ischemia through knocking down AIBP activity in the cardiac muscle fundamentally to solve the myocardial microcirculation disturbance problem for many patients suffering ischemic heart disease and end-stage heart failure, so as to improve the micro-environment for survival of myocardial cells and supply of nutritional energy matters and give a certain theoretical foundation for completely curing ischemic heart disease. Objectives1. To discuss rat CMECs separation, cultivation, and verification in vitro, and lentiviral transfection, screen and reduce the expression of AIBP gene.2. To observe the influence on reducing the expression of AIBP gene to vascular regeneration function of rat CMECs.3. To research the influence on reducing the expression of AIBP gene to coronary microvascular regeneration after the rat suffering myocardial infarction. Methods1. Rat CMECs separation, cultivation, verification in vitro and lentiviral transfectionCombining mixed enzymatic digestion method with differential adhesion method we separate SD rat CMECs, and adopted Dil-Ac-LDL phagocytosis detecting method to make the identification of it, and then use P2- P3 CMECs to carry out AIBP sh RNA transfection via lentivirus mediation to reduce the expression of AIBP gene in CMECs. RT-PCR and Western Blot was to detect the expressing level of AIBP gene and protein in normal and transfected cells, and screen out the gene sequence that AIBP expression was effectively reduced.2. Observing the influence on vascular regeneration function of rat CMECs by reducing the expression of AIBP geneWe use P2- P3 CMECs to carry out AIBP gene reducing and transfection. Compared control group with L-NC group, we use Western Blot to detect the VEGFR2 expression and p AKT protein level in cells, then Alama Blue colorimetric method to detect cell reproductive capacity, Transwell cell migration experiment to detect cell migration capacity, at last Matrigel matrix rubber-hose structural formation experiment to detect vascular regeneration capacity of CMECs.3. The influence on rat coronary microvascular regeneration by reducing the expression of AIBP gene after myocardial infarctionEstablished rat’s myocardial infarction model and injected lentivirus at the place of myocardial infarction, adopted Western Blot to detect the expression level of AIBP protein in normal and transfected cardiac muscle, adopted immunohistochemical method to detect the expression level of CD31, adopted Masson trichrome to detect the degree of myocardial fibrosis, and adopted cardiac ultrasonography to detect the cardiac function. Results1. The rat’s CMECs was successfully separated and verified. After 5 d- 6 d cultivating, the mature cardiac microvascular endothelial cells were closely integrated and linked together and showed a polygon-type “appearance as paving stone”, and the phagocytic function of Ac-LDL was normal. Through mutual verification of western Blot detection result and RT-PCR, the gene sequence that AIBP expression was reduced effectively was screened out successfully.2. The experimental result in vitro showed that the level of VEGFR2 expression and AKT phosphorylation on CMECs increased after AIBP expression was knocked down. It might improve the proliferation capacity, cell migration capacity and pipe formation capacity of CMECs(P < 0.01).3. The animal experimental result showed that the amount of myocardial microvessel regeneration was obviously increased in the area of myocardial infarction after AIBP expression was blocked, and the degree of myocardial fibrosis was obviously decreased and the rat’s left ventricular function was improved obviously(P < 0.01). ConclusionKnocking down the expression of myocardial vascular AIBP gene could promote the myocardial microvessel regeneration obviously. The CMECs regeneration capacity in vitro can be promoted by knocking down the expression of CMECs AIBP gene. Knocking down the expression of AIBP gene in rat’s cardiac tissue could promote the coronary microvessel regeneration obviously, and cardiac function was improved. Promoting the generation and neogenesis of myocardial microvessel is important to cure ischemic heart disease. Perhaps it is more valuable prospect to clinical treatment of myocardial infarction.